Biomolecule Separation and Analysis
on HPLC Columns
An Trinh, Michael Ye, Tom Rutkoski, Yizhu Guo, Bill Maule, and Hillel Brandes
Bioseparations Group, Supelco, Supelco Park, Bellefonte, PA, 16823-0048, USA
Phone: 800-359-3041 or 814-359-3041; or Fax 800-359-5468 or 814-359-3044; or Email [email protected]
©2002 Sigma-Aldrich Co.
T402058
ETO
Abstract
Recent advances in the “post-genomic” era of proteomics and biotechnology have put research
on the path towards further understanding the cellular/ biological nature of diseases. By controlling disease through an acute understanding of our own body’s defense mechanisms, bio-engineered drugs, peptide therapeutics, and biotechnology represent the promise of new medical
treatments for the new millennium.
Whether it is resolving a tryptic digest for a peptide map, assessing the purity and recovery of a
downstream bioprocess, or developing a preparative LC method, it becomes critically clear of the
need for innovative high quality separation technology in all areas of bio-pharmaceutical research.
As a result, Supelco has introduced its newest line to the Discovery HPLC column family —
Discovery BIO Wide Pore (300Å).
In our presentation, we will show an array of peptide/ protein applications on reversed-phase
HPLC demonstrating key aspects imperative to the separation, analysis, and purification of biomolecules. Such studies include: peptide resolution in low TFA mobile phase environments; increased bonding stability of C5 over C4 alkyl functional groups; the importance of matched selectivity across particle sizes; and differences in selectivity in relation to solid phase chemistry.
Introduction
Contemporary advances in understanding the cellular and biological nature of diseases has put
us on the path of controlling disease using our own body’s natural defense mechanisms. As a
result, bioengineered drugs, peptide therapeutics, and biotechnology represent the promise of
new medical treatments.
When analyzing or isolating biomolecules using HPLC technology, the most important factor to
consider when developing a method is RESOLUTION. This is crucial for analytical assessment
and isolation of biological samples. The most powerful attribute of reversed-phase LC technology
relative to other chromatographic mediums is its resolving power. As a result, reversed phase
HPLC plays an increasingly critical role in such areas as analytical biomolecule characterization,
proteomics, semi-preparative and preparative purification, and downstream process monitoring.
In this report we will discuss key performance aspects regarding reversed-phase LC technology in
the separation, analysis, and purification of biomolecules through application examples representative of today’s biomolecular research.
The Resolution of Structurally Similar Biomolecules
Using Reversed-Phase HPLC
Resolution is one of the most important factors to consider when analyzing or isolating structurally similar biological molecules via HPLC. For example, insulin is composed of two peptide
chains: a 21 amino acid A chain and a B chain of 30 amino acids. The two chains are linked via
two disulfide bonds, and much of the sequence is conserved across species (including the locations of the disulfide bonds) allowing for a very similar three dimensional conformation regardless
of species types. Porcine insulin differs from human by only one amino acid, and bovine differs by
three amino acids. In Figure 1, we show the separation of these three insulin types using 300Å C5
reversed-phase HPLC column.
Angiotensin is a peptide that is associated with circulatory disorders. Human angiotensins I, II
and III contain a seven amino sequence conserved between the three types. The three peptides
differ only by three amino acid residues. Angiotensin II differs from angiontensin II by an N-terminal asparate; and angiotensin I contains an N-terminal asparate as well as two C-terminal amino
acids (histidine and leucine). At low pH conditions (most typical for peptide chromatography), angiotensins II & III are not resolved since the neutral aspartate residue contributes no retention.
Therefore, resolution of the two angiotensins is typically conducted at alkaline pH. In this application, we were able to achieve baseline resolution of the three peptide types using a 300Å C8 reversed-phase HPLC column at neutral pH (Figure 2).
Figure 1: Insulin from Various
Species Separated on a
Discovery BIO Wide Pore C5 Column
Column:
Discovery BIO Wide Pore C5,
15cm x 4.6mm, 5µm
(A) 71:29, (0.1% TFA in water):(0.1% TFA in
CH3CN);
(B) 68:32, (0.1% TFA in water):(0.1% TFA in
CH3CN)
1.0mL/min
ambient
215nm
5µL each at 1mg/mL in 0.1%TFA
0-100%B in 30 min
Mobile Phase:
Flow Rate:
Temp.:
Detection:
Injection:
Gradient:
Figure 2: Resolution of Angiotensins
at Neutral pH Using
Discovery BIO Wide Pore C8 Column
Column:
Mobile Phase:
Flow Rate:
Temp.:
Detection:
Injection:
Gradient:
1. Bovine insulin
2. Human insulin
3. Porcine insulin
1
2
2
4
6
Min
1. Angiotensin II (1.67g/L)
2. Angiotensin III (1.67g/L)
3. Angiotensin I (1.67g/L)
1
2
3
0
Discovery BIO Wide Pore C8,
15cm x 4.6mm, 5µm
(A) 10mM NH4H2PO4/NH4OH, pH 7
(B) 50:50, 20mM NH4H2PO4/NH4OH, pH 7:MeCN
1mL/min
30°C
215nm
6µL in water
30-60% B in 15 min
8
10
0
G001580
(DRVYIHPF)
(RVYIHPF)
(DRVYIHPFHL)
3
10
Min
20
G001503
Resolving Target Proteins from Degradation Products and Impurities
Proteins and polypeptides are subject to various types of molecular transformations that affect
their biological activity and integrity. Adequate resolution of target biomolecules from their degradation products and impurities becomes crucial in protein and peptide analyses. This is especially important when assessing the yield and purity of a downstream processing step or a
benchtop protein isolation procedure. With the appropriate methodology, reversed-phase HPLC
can provide the researcher with the resolution required to handle such studies.
In this application, we achieved baseline resolution using a 300Å C5 reversed-phase HPLC column when analyzing six hydrophobic proteins (Figure 3). Note the separation that was achieved
for lysozyme and myoglobin and their relative impurities.
Figure 3: Separation of Proteins on Discovery BIO Wide Pore C5
Column:
Mobile Phase:
Flow Rate:
Temp.:
Detection:
Injection:
Gradient:
Discovery BIO Wide Pore C5, 15cm x 4.6mm, 5µm
(A) 75:25, (0.1% TFA in water):(0.1% TFA in CH3CN);
(B) 25:75, (0.1% TFA in water):(0.1% TFA in CH3CN)
1.0mL/min
ambient
220nm
12µL in 0.1%TFA
0-100%B in 25 min
Impurity in
Lysozyme
RNase (13.7kDa, 1mg/mL)
Cytochrome c (12.4kDa, 1mg/mL)
Lysozyme (14.3kDa, 1mg/mL)
BSA (67.0kDa, 2.5mg/mL)
Myoglobin (18.8kDa, 1mg/mL)
Ovalbumin (45.3kDa, 3.5mg/mL)
3
2
600
1.
2.
3.
4.
5.
6.
4
Impurity in Myoglobin
5
1
6
400
200
0
0
10
Min
20
G001488
Proteolytic Digestions & Reversed-Phase HPLC
As we enter the post-genomic era, more emphasis is being placed on identifying and characterizing all proteins expressed by a cell or tissue under normal and perturbed states. This gave birth to
a new era of study and thought- Proteomics. As more and more complicated protein/peptide functions become elucidated, the promise of new medical treatments approaches as more and more
relevant drug/diagnostic targets can be determined.
Within the field of proteomics, peptide maps have become a mainstream tool for elucidating protein structure, sequence and purity. In such studies, a protein of interest is subjected to digestion
resolved using LC technology. Because a typical digestion can yield dozens to hundreds of peptide fragments, adequate resolution is essential for analyzing these complex samples.
In this application, we resolved 74 peptides generated from a carboxymethylated apohemoglobin
tryptic digest using a 300Å C18 reversed-phase HPLC column (Figure 4).
Figure 4: Tryptic Digest of Carboxymethylated Apohemoglobin
on a Discovery Wide Pore C18
Column:
Mobile Phase:
Flow Rate:
Temp.:
Detection:
Injection:
Gradient:
Discovery BIO Wide Pore C18, 15cm x 4.6mm, 5µm
(A) 95:5, (0.1% TFA in water):(0.1% TFA in CH3CN);
(B) 50:50, (0.1% TFA in water):(0.1% TFA in CH3CN)
1.0mL/min
30°C
215nm
50µL carboxymethylated apohemoglobin tryptic digest in 50mM NH4HCO3
0-100%B in 65 min
Discovery BIO Wide Pore C18
74 peptides resolved
0
20
40
Min
60
G001510
Comparison: Separation of a Proteolytic Digest using C5 vs. C8 vs. C18
Bonded phase chemistry dictates many aspects of the separation from retention to resolution.
In this study, we compared the effects of carbon chain length (C5, C8 & C18) of the bonded
phase for the separation of a carboxymethylated apohemoglobin tryptic digest (Figure 5). The
three different bonded phases display different selectivity towards the peptides as observed in
their elution profiles. This is especially notable in the 10 minute region for the 300Å C18
reversed-phase HPLC column.
Figure 5: Each Discovery BIO Wide Pore Phase
Gives Unique Elution Profiles of
Carboxymethylated Apohemoglobin
Peptide Fragments
Columns:
Mobile Phase:
Flow Rate:
Temp:
Detection:
Injection:
Gradient:
(A) Discovery BIO Wide Pore C5
(B) Discovery BIO Wide Pore C8
or (C) Discovery BIO Wide Pore C18,
each 15cm x 4.6mm, 5µm
(A) 95:5, (0.1% TFA in water):
(0.1% TFA in CH3CN);
(B) 50:50, (0.1% TFA in water):
(0.1% TFA in CH3CN)
1.0mL/min
30°C
215nm
50µL carboxymethylated apohemoglobin
tryptic digest in 50mM NH4HCO3
0-100%B in 65 min
Discovery BIO
Wide Pore C5
Note the different
elution patterns
between the
Discovery BIO
Wide Pore phases.
G001506
0
20
40
60
Min
Discovery BIO
Wide Pore C8
0
20
40
60
Discovery BIO
Wide Pore C8
shows better
resolution in
this region.
G001504
Min
Discovery BIO
Wide Pore C18
0
20
40
Min
60
G001505
Reversed-Phase HPLC Column Stability & Reproducibility
To ensure that the methods described in this report will be reproducible, we tested the columns
used in this study for pH stability and column bleed (MS detection). Trifluoroacetic acid (TFA) at
pH 2 is a commonly used mobile phase in RP-HPLC separation of proteins and peptides. A robust
method dictates that the column is stable under these harsh conditions. In this study, a TFA mobile phase at 70°C was passed through the 300Å C18 reversed-phase HPLC column used in this
report. After 40,000 bed volumes, selectivity and peak shape remained essentially unchanged
(Figure 6). A similar study was conducted using an akaline pH mobile phase of 11.5 (Figure 7). Retention time of the molecular probes remained stable after 40,000 bed volumes.
Short chain alkyl bonded phase such as C3 and C4 are routinely used for RP-HPLC separations of
proteins and hydrophobic peptides. However, both C3 and C4 phases hydrolyze at low and high
pH resulting in short column life and poor reproducibility. In this study, we compare the stability
of a C5 phase vs. a conventional C4 phase when employed with a TFA mobile phase (Figure 8).
The results indicate by adding one C atom to the bonded chain length, marked improvements
were obtained in terms of stable peak shape and efficiency.
LC/MS applications are particularly sensitive to bleed ions potentially generated from the analytical columns. The presence of overlapping bleed ions can obscure the presence of peaks of interest in total ion chromatogram analyses resulting in a decrease in sensitivity.
In this study, we performed an LC/MS analysis of blank injections run with and without the presence of our 300Å C18 reversed-phase HPLC column under gradient conditions. Total ion chromatography and mass spectrometry data were compared to determine the presence of bleed ions
that may have been generated from the analytical columns (Figure 9). Essentially no m/z peaks
were generated 300Å C18 reversed-phase HPLC column used in this report.
Figure 6: Stability of Discovery BIO
Wide Pore C18 at pH 2 and 70°C
Column:
Mobile Phase:
Flow Rate:
Temp.:
Detection:
Injection:
Gradient:
Figure 7: Discovery BIO Wide Pore
C18 Stability at pH 11.5
Discovery BIO Wide Pore C18, 5cm x 4.6mm, 5µm
(A) 5:95, (0.5% TFA in water):(0.5% TFA in CH3CN);
(B) 25:75, (0.5% TFA in water):(0.5% TFA in CH3CN)
2.0mL/min
70°C
220nm
5µL, 2.5µg each peptide
(Sigma Peptide Mix, Cat. No. H 2016)
in mobile phase A
2-24%B in 22 min, 8 min at 100%A
1.
2.
3.
4.
5.
1
1
2
2
Column:
Mobile Phase:
Flow Rate:
Temp.:
Discovery BIO Wide Pore C18,
5cm x 4.6mm, 5µm
65:35, 50mM pyrrolidine HCl
(pH 11.5):CH3CN
2.0mL/min
35°C
1.
2.
3.
4.
Pindolol
N-Methylaniline
Propranolol
Toluene
Stable retention on Discovery BIO Wide Pore
C18 after 40,000 column volumes at pH 11.5.
Gly-Tyr
Val-Tyr-Val
Met-Enkephalin
Leu-Enkephalin
Angiotensin II
3
3
5
4
4
5
Final Injection
Initial Injection
0
10
Min
20
G001562, 63
Note: Stability was measured using small molecule probes because they are
generally more sensitive to changes in the silica and bonded phase chemistry
than peptides and proteins. If the retention and selectivity for the small molecule probes does not change, it is very likely that the protein or peptide
separations will be stable as well.
G001586
Figure 8: Comparison of Low pH Stability of Discovery BIO Wide Pore C5
versus a Conventional C4 Column
Mobile Phase:
Flow Rate:
Temp.:
Detection:
Injection:
Gradient:
Discovery BIO Wide Pore C5, 5cm x 4.6mm, 5µm
or (B) Conventional protein and peptide C4, 15cm x 4.6mm, 300Å, 5µm
(A) 5:95, (0.5% TFA in water):(0.5% TFA in CH3CN);
(B) 25:75, (0.5% TFA in water):(0.5% TFA in CH3CN)
2.0mL/min
30°C
220nm
5µL, 2.5µg each peptide (Sigma Peptide Mix, Cat. No. H 2016) in mobile phase A
2-24%B in 22 min, 8 min at 100%A
Efficiency on Discovery BIO Wide Pore C5 is stable even
after 25,000 column volumes (222 gradient cycles).
Efficiency: % of first injection
Columns:
Under the same
conditions, there
was significant loss
of efficiency on
conventional C4.
G001588
Figure 9: Undetectable LC/MS Bleed on Discovery BIO Wide Pore C18 Column
Column:
Mobile Phase:
Flow Rate:
Temp.:
Gradient:
Discovery BIO Wide Pore C18, 15cm x 4.6mm, 3µm
(A) 0.1% TFA in water; (B) 0.1% TFA in CH3OH
1.0mL/min
30°C
0-100%B in 15 min, 100%B for 5 min, 0%B for 10 min
No TIC baseline rise relative
to blank run (gradient run
without column)
Blank Run
(Baseline offset due
to lack of column)
100
Discovery BIO
Wide Pore C18
M/S Area
%
0
Essentially no m/z
peaks generated from
Discovery BIO Wide Pore C18
compared to a blank run.
“X” indicates peaks that were
also seen in the blank run.
0
10
20
30 Min
G001522
M/S Spectrum (shaded area)
G001522
Scalability of the Separation
Shorter and narrower ID columns allow for more rapid analysis and better LC/MS applicability. 3
and 5µm particle size stationary phases provide maximum resolution for complex protein and peptide mixtures. 10µm in large column dimensions are ideal for purifying mg to gram amounts of
proteins for further analyses. In order to determine if the columns employed are scalable from
analytical to prep for these diverse purposes and applications, we tested the 300Å C18 phase for
matched selectivity across three particle sizes (3, 5 and 10µm) and two column dimensions using
a standard peptide mix (9 peptides) (Figure 10). With the proper scaling factors, matched selectivity was observed from analytical to prep using the 300Å C18 phase employed in this report.
Figure 10: Matched Selectivity from Analytical to Preparative on
Discovery BIO Wide Pore C18
Column:
Mobile Phase:
Linear Velocity:
Temp.:
Detection:
Sample:
Discovery BIO Wide Pore C18, 15cm x 4.6mm, 3µm
Discovery BIO Wide Pore C18, 15cm x 4.6mm, 5µm
Discovery BIO Wide Pore C18, 15cm x 10mm, 10µm
(A) 80:20, (0.1% TFA in Water):(0.1% TFA in CH3CN),
(B) 66:34, (0.1% TFA in Water):(0.1% TFA in CH3CN)
6.02cm/min
30°C
215nm
Sigma Peptide Mix (Sigma Cat. No. P 2693) in 0.1% TFA
Column Parameters & Run Conditions:
Column
Column Volume(mL)
15cm x 4.6mm, 3µm
1.64
15cm x 4.6mm, 5µm
1.71
15cm x 10mm, 10µm
8.01
Gradient:
Column Volumes
0
2
9
%A
100
100
0
%B
0
0
100
Injection (µL)
5.0
5.0
24.5
3µm
Discovery BIO Wide Pore C18
15cm x 4.6mm, 3µm
5µm
Discovery BIO Wide Pore C18
15cm x 4.6mm, 5µm
10µm
Discovery BIO Wide Pore C18
15cm x 10mm, 10µm
Flow (mL/min)
1.00
1.00
4.73
0
2
4
6
8
10
Min
12
14
16
G001512, 13, 11
Proteomics & Capillary LC Chromatography Interfaced with Mass Spectrometry
A major trend in proteomics research today is the need for detecting very low levels of proteins
and peptides (pmol level) from small volumes (low micro-liter range) of samples. Capillary and
microbore LC technology offer the researcher a tool that specifically addresses this growing need.
Because samples are diluted over a small column volume, capillary and microbore columns offer
greater efficiency and sensitivity than columns with conventional internal diameters (e.g., 4.6mm).
When interfaced with an MS detection unit, capillary/microbore columns help provide structural information on proteins and peptides at extremely low copy numbers in the cell.
In this study, we analyzed 500pmol (5µL) of ß-lactoglobulin tryptic digest using a 300Å C18 reversed phase capillary HPLC column (15cm x 0.5mm) (Figure 11); and 50pmol of angiotensins I, II
and III in water using a 300Å C18 reversed phase capillary HPLC column (15cm x 0.32mm) (Figure
12). Note that an increase sensitivity of up to 84 and 207 times can be achieved using a 0.5mm
and 0.32mm ID column respectively over standard 4.6mm ID dimensions.
Figure 11: ß-Lactoglobulin Tryptic
Digest on 0.5mm ID Discovery BIO
Wide Pore C18 Capillary
Figure 12: Angiotensins on 0.32mm ID
Discovery BIO Wide Pore C18 Capillary
Column:
Column:
Discovery BIO Wide Pore C18, 15cm x
0.5mm, 5µm
Mobile Phase: (A) 0.1% TFA in water;
(B) 0.1% TFA in CH3CN
Flow Rate: 14µL/min
Temp.: 30°C
Injection: 500pmol (5µL) ß-Lactoglobulin tryptic
digest in 50mM NH4HCO3
Gradient: 5-40%B in 70 min
MS conditions: +ESI mode Capillary Temp 130°C, Source
Voltage 2.5KV, Capillary Voltage 12V
17.17
337.3
50
15.90
458.9
27.17
1044.6
12.36
573.3
30.49 34.37
772.1 522.5
36.66
322.0
35.06
522.6
24.54
775.4
37.71
522.6
0
0
10
20
Min
30
4.10
931.5
III
50
0
0
40
G001591
60
40
20
Relative Abundance
18.46
623.4
3.34
8.36
1046.6 649.1
100
II I
Relative Abundance
100
Relative Abundance
27.66
22.80 859.2
21.42 818.7
29.17
696.2
555.2
21.00
419.4
Discovery BIO Wide Pore C18,
10cm x 0.32mm, 3µm
Mobile Phase: (A) 65:35, (10mM NH4OAc, pH 7):(50% CH3CN in
20mM NH4OAc, pH 7)
(B) 25:75, (10mM NH4OAc, pH 7):(50% CH3CN in
20mM NH4OAc, pH 7)
Flow Rate: 6µL/min
Temp.: ambient
Injection: 50pmol in water
Gradient: 0-100%B in 12.5 min
MS conditions: +ESI mode Capillary Temp 130°C, Source
Voltage 2.5KV, Capillary Voltage 12V
10
Min
20
40
524.0
1046.6
611.3 1047.5
784.4
466.5 931.5
20
669.4
100
MS confirms
identity of
peaks.
649.1
50
0
400
800
m/z
1200
G001592
Conclusion
As we move further into the 21st century, we rely on the biopharmaceutical industry to help fight
disease with new biotherapeutic agent and to increase our understanding of aging and disease
through proteomics, the cataloging of proteins expressed by the genome. Reversed-phase HPLC
technology will be an important analytical tool in characterizing, elucidating, and purifying key
biological molecules.
In our report, we discussed some of the key challenges these researchers face when
incorporating RP-HPLC technology in their methodology. These issues include:
●
●
●
●
●
●
●
The resolution of structurally similar proteins and peptides
Resolving target biomolecules from their degradation products and impurities
The analysis of protelytic digestions using RP-HPLC technology
Selectivity differences between three different carbon chain length bonded phases
Method and column stability and reproducibility
Equivalent performance selectivity during scaling
Proteomics and Capillary/Microbore LC technology